| Objective: Human immunodeficiency virus(HIV)is still widely prevalent in the world and has a serious impact on human life,health and social development.Preventive vaccines are the most effective means to combat the epidemic of HIV-1.Throughout the history of HIV-1 vaccine development,the first and second generation vaccines targeted at humoral and cellular immunity ended in failure.The phase III clinical trial of the third generation vaccine RV144,which focuses on comprehensive immunity,only detected 31% of the protection efficiency,suggesting that at present,we still have not clear understanding of the mechanism of how b NAbs(Broadly neutralizing antibodies,b NAbs)is induced in HIV-1 infected people,There are still great challenges in vaccine design and monoclonal antibody treatment.Therefore,it is very important to analyze the mechanism of b NAbs production in HIV-1 infected persons and their coevolution with the virus.HIV-1 infection leads to serious defects in humoral response,mainly manifested by the polyclonal activation of B cells and the abnormal distribution of B cell number and subgroup.HIV-1 infection also affects the auxiliary effect of T cells on B cells,resulting in damage to the formation of germinal centers,thus affecting the further somatic mutation and affinity maturation of early autoneutralizing antibodies,resulting in the development of b NAbs.Research shows that after 2-3 years of co-evolution of virus and antibody,only 20% to 30% of HIV-1 infected people can produce antibodies with broad-spectrum neutralizing ability in their serum.It is known that many HIV-1 b NAbs have typical genetic and structural characteristics,such as auto-responsiveness or multi-reactivity,preference use of heavy chain V gene,high frequency somatic cell hypermutation and long heavy chain CDR3(CDR3)sequence,and BCRs expressing these characteristics are very rare in the B cell bank of healthy people or HIV-1 infected people without broad-spectrum neutralizing activity,It is suggested that HIV-1 infected persons with broad-spectrum neutralizing activity may have a unique B cell evolution process leading to the generation of b NAbs.A clear understanding of the response,development pathway and BCR evolution characteristics of B cells in HIV-1 infected individuals with broad-spectrum neutralizing activity will help guide the rationaldesign of HIV-1 vaccines to trigger similar reactions.Due to the high variability of HIV-1 virus,people with different HIV-1 broadspectrum neutralization activities are stimulated by different antigens,and the formation mechanism of broad-spectrum neutralization response in vivo is different.International HIV-1 virus and antibody co-evolution teams generally use a variety of virus and antibody analysis methods to analyze the differentiation and development of B cells and the occurrence and development of antibodies in individuals.The sequencing technology of single-cell Immune repertoire realizes the joint analysis of transcriptome and Immune repertoire library at the single cell level,which can more comprehensively analyze the immune response of HIV-1 infected B cells,and provides a new direction for revealing the internal factors of broad-spectrum neutralization activity.In this study,we used single cell transcriptome and immunohistochemical library sequencing technology to conduct a combined analysis of single cell transcriptome and BCR library on the peripheral blood B cells of an HIV-infected person(320975)with broad-spectrum neutralizing activity in plasma for 3.38 years.Through the analysis of BCR progression characteristics and B cell response related to broad-spectrum neutralizing activity,and combining with the evolution track of 320975 virus,we can comprehensively analyze the possible mechanism of 320975 broadspectrum neutralizing activity,Provide more information for HIV-1 vaccine design.Methods:1.The broad-spectrum neutralizer 320975 is CRF01_The ML Tree evolution analysis and Highlighter visualization analysis of the patients with super-infection in the AE subtype suggest that the time of super-infection is 0.12 to 0.79 years.The viruses in the body are divided into two clusters of dominant and inferior strains that are continuously under the pressure of immune selection.The plasma heterogenous neutralization test indicates that the patient has obtained broad-spectrum neutralization activity in 1.98 and the plasma neutralization ability has reached its peak,which can neutralize 58% of the heterologous global panel virus.2.The data analysis of the single cell immunohistochemistry library showed that the proportion of BCR sequences with the length of heavy chain CDR3>19aa in infected persons 320975 was 26%,and the utilization rate of IGHV4-34 in the heavy chainIGHV gene was the highest,and the IGHV4-34 sequence alignment results showed that the IGHV4-34 sequence containing two amino acid motifs representing autoreactivity,AMY and NHS,accounted for the majority;Through the joint analysis of 320975 single cell transcriptome data,we identified two cell subpopulations of na? ve B cells and memory B cells;The differential expression analysis of common broadspectrum neutralizing antibody using IGHV gene found that IGHV4-34 gene was highly expressed in ITGAX memory B cells related to autoreactivity.The results of single cell transcriptome and immunohistochemistry library showed that 320975 was in a state that allowed the existence of self-reactive clones.3.The evolution analysis of 320975 antibody heavy chain and the use of the same V region pedigree gene shows that most of the 320975 antibodies in vivo have different evolution patterns from the known broad-spectrum neutralizing antibodies,but there may be antibody sequences similar to the broad-spectrum neutralizing antibodies 4022 targeting V3 region and VRC-CH32 targeting CD4 bs.The validation analysis of the epitopes of the representative broad-spectrum neutralizing monoclonal antibody found that the virus was subjected to continuous antibody selection pressure in the V3 region and CD4 bs,and was not caused by the virus recombination within the subtype.The BCR evolution analysis and the epitope analysis of the representative monoclonal antibody jointly suggested that there was an immune selection pressure targeting the V3 region and CD4 bs in 320975.4.Six memory B clusters were identified by dimensionality reduction clustering of 320975 memory B cells.The results of pseudo-time analysis showed that six memory B clusters were at different stages of differentiation and development,memory B cluster 0 is at the starting point of differentiation and development,memory B cluster 2 and memory B cluster 3 is in the middle stage of development,while memory B cluster 1、memory B cluster 4 and memory B cluster are at three different ends of development.The difference analysis of the expression of heavy chain VH gene of 3209756 known broad-spectrum neutralizing antibodies in memory B cluster showed that the VH gene of most known broad-spectrum neutralizing antibodies,including IGHV1-2 and IGHV5-51,was significantly enriched in the cells of memory B cluster 2 and memory B cluster 3,and the GO enrichment results showed that memory Bcluster 2 was enriched in the ribosome-related pathway,while memory B cluster 3 was enriched in the B-cell activation-related pathway,It is suggested that this group of cells may be related to the formation of 320975 broad-spectrum neutralization activity.Results:1.Broad-spectrum neutralizer 320975 is a case of CRF01_AE super-infected patients,the evolution analysis suggested that the super-infection had occurred in 0.79,and the plasma heterogenous neutralization results suggested that the broad-spectrum neutralization activity was obtained in 1.98,and the plasma neutralization ability reached the peak,which could neutralize 58% of the allogenic Global panel virus2.The data analysis of the single cell Immune repertoire library indicates that 320975 has antibodies with long CDR3 heavy chain,which may be induced by glycandependent epitopes.Through the cluster and annotation of the combined analysis of the single cell transcriptome data,we identified two cell types of na?ve B cells and memory B cells.Among them,the differential expression analysis of the IGHV gene used by the common broad-spectrum neutralizing antibody found that,IGHV4-34 gene is highly expressed in B cell clusters related to autoreactivity.3.The evolution analysis of 320975 antibody heavy chain and the use of the same V region pedigree gene shows that most of the 320975 antibodies in vivo have different evolution patterns from the known broad-spectrum neutralizing antibodies,but there may be antibody sequences similar to the broad-spectrum neutralizing antibodies 4022 targeting V3 region and VRC-CH32 targeting CD4 bs.The mutation analysis of the representative monoclonal antibody epitopes of the virus found that the virus was subjected to continuous antibody selection pressure in V3 region and CD4 bs,and the point mutation was not caused by the virus recombination within the subtype.The BCR evolution analysis and the representative monoclonal antibody epitope analysis jointly suggested that there might be immune selection pressure targeting V3 region and CD4 bs in 320975.4.Six memory B clusters were identified by dimensionality reduction cluster analysis of 320975 memory.The results showed that six memory B clusters were at different stages of differentiation and development,memory B cluster 0 is at the starting point of differentiation and development,memory B cluster 2 and memory B cluster 3 is inthe middle stage of development,while memory B cluster 1,memory B cluster 4 and memory B cluster 5 is at three different ends of development.The differential analysis of 320975 known broad-spectrum neutralizing antibodies using heavy chain VHF gene expression of 6 memory B clusters showed that most of the known broad-spectrum neutralizing antibodies using VH genes,including high VH1-2 and high VH5-51,were significantly enriched in memory B cluster 2 and memory B cluster 3 cells.The results of the deconcentration showed that memory B cluster 2 was enriched in the ribosomerelated pathway,while memory B cluster 3 was enriched in the B-cell activation-related pathway,Combined with the mutation of virus monoclonal antibody and the evolution analysis of BCR company,the results showed that the memory B cluster 2 and memory B cluster 3 cells might be related to the formation of 320975 broad-spectrum neutralization activity.Conclusion:1.320975 with broad-spectrum plasma neutralizing activity and CRF01_AE subtype superinfection,plasma broad-spectrum neutralizing activity was formed 1.98 years after initial infection.2.320975 has long CDRH3 in its body.Most of the IGHV4-34 sequences have autoreactive amino acid motifs and IGHV4-34 is highly expressed in the memory B cell cluster related to autoreactivity,which indicates that 320975 is in the immune permissive state of autoreactivity.3.The combination of antibody evolution and virus evolution suggests that the broadspectrum neutralization activity of 320975 may target V3 and CD4 bs regions.4.Memory B cluster 3 is significantly enriched in B cell activation-related pathways,which may be related to the formation of broad-spectrum neutralization activity. |
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